how they work
Because SYTOX® dyes do not cross intact cell membranes and do increase fluorescence upon dsDNA binding, they are among our most brilliant dead cell stains. Dead cells can be easily distinguished from live cells by flow cytometry by measuring the increased fluorescence signal due to dsDNA binding. SYTOX® Dead Cell Stains can be applied to cells and visualized without an additional wash step because they are nonfluorescent in aqueous media.

A mixture of heat-killed and live Jurkat cells was stained with 30 nM SYTOX® Green Dead Cell Stain and incubated at room temperature for 20 min. Cells were analyzed on the Attune® Acoustic Focusing Cytometer equipped with a 488 nm laser. Fluorescence emission was collected using a 530/30 bandpass filter. Live cells are easily distinguished from the brighter dead cell population.

what they are
ABfinity™ recombinant rabbit monoclonal antibodies are available for many targets, including JAK1 [phospho Y1022], STAT1 [phospho Y701], STAT4, and STAT6 [phospho Y641]. JAK-STAT signaling is important in regulating immune responses and autoimmune diseases, especially when interferon-γ signaling is compromised in various viral infections.

what they offer

High sensitivity and specificity

Excellent lot-to-lot consistency

Extensive validation and characterization

how they work
ABfinity™ recombinant rabbit monoclonal antibodies are produced from specific recombinant clones. ABfinity™ technology helps to ensure consistent antibody performance so you don’t have to reoptimize your assay with each new lot.

Cells were stimulated with 100 ng/mL interferon-γ (top right) or unstimulated (top left) and labeled with rabbit anti-STAT1 [pY701] (1 µg/mL). Stimulated cells were preincubated with the phosphopeptide used as an immunogen (bottom right) or with nonphosphopeptide (bottom left), before incubating with Alexa Fluor® 488 goat anti–rabbit IgG secondary antibody at 1:1,000 dilution. The signal is prevented by incubation with the phosphopeptide but not with the nonphosphopeptide, demonstrating phosphospecifity of the antibody.

NEW APPLICATIONS

Visualizing Endocytosis

Lysosomally directed and recycling endosomal pathways are critical for a variety of cellular functions. Labeled and unlabeled low-density lipoprotein (LDL), transferrin, lipid dyes such as FM® lipophilic styryl dyes and lipophilic tracers (Dil, DiO, DiD, DiA, and DiR), and pHrodo™ indicators are valuable tools for interrogating endocytic pathways. In a recent study, Boucrot et al. [1] used several of these tools to determine the importance of the AP2 protein in the mechanism of endocytosis.

Transferrin Conjugates
Transferrin is a monomeric serum glycoprotein that binds up to two Fe3+ atoms for delivery to vertebrate cells through receptor-mediated endocytosis. Fluorescently labeled transferrin has been used to measure transferrin receptor-binding affinity in mammals and parasites, to investigate endocytosis, and to study endocytic recycling pathways. Alexa Fluor® transferrin conjugates are highly recommended because of their brightness, enhanced photostability, and lack of sensitivity to pH.

Fluorescent Low-Density Lipoprotein Complexes
The human LDL complex delivers cholesterol to cells by receptor-mediated endocytosis. In addition to unlabeled LDL, which has been reported to be an effective vehicle for selectively delivering antitumor drugs to cancer cells, we offer two classes of labeled LDL probes—those containing an unmodified apoprotein, used to study the mechanisms of normal cholesterol delivery and internalization, and those with an acetylated apoprotein, used to study endothelial, microglial, and other cell types that express receptors that specifically bind this modified LDL.

Lipid Dyes
Lipid dyes are useful as general-purpose probes for investigating endocytosis and for simply identifying cell membrane boundaries. We offer FM® lipophilic styryl dyes and lipophilic tracers (Dil, DiO, DiD, DiA, and DiR), which fluoresce brightly upon integration into cellular membranes. Both fixable and unfixable dyes are available in various colors.
pHrodo™ Indicators
The pH-sensitive pHrodo™ dye is nonfluorescent at neutral pH and fluoresces bright red in acidic environments. This increase in fluorescence signal at low pH makes it ideal for studying endocytosis and its regulation by drugs and/or environmental factors. The unique pHrodo™ dye–based system measures phagocytosis and endocytosis based on acidification of the particle or protein conjugates as they are ingested by cells.

PROVEN PERFORMERS

Application Notes for the Countess® Automated Cell Counter

The Countess® Automated Cell Counter counts live and dead cells and measures viability, typically in just 30 seconds. For specific protocols and technical information, you can download a variety of application notes relating to your research area. Topics include:

DEPARTMENTS

Buzzworthy

High-throughput screening platforms to analyze anticancer treatments and other therapies are valuable tools in the modern clinical research setting. A recent publication from Wlodkowic and colleagues surveys a few of the patented DNA-binding SYTO® dyes for the detection and tracking of apoptosis in diverse cell lines and in primary tumor samples. Loss of apoptosis is one of the defining parameters of malignancy, and the mode of action of some of the SYTO® dyes makes them particularly suited for monitoring this cellular process. Wlodkowic et al. cite the broad range of visible excitation and emission, high quantum yield upon binding to nucleic acids, low cost, and low toxicity profiles as beneficial in their investigations. They speculate that these probes will be useful as a first-line screening agent for novel therapeutics and that they could be very valuable in multiplexed assays and in microfluidics devices.

The View

Transverse Section of Fixed Zebrafish Retina. A transverse section of fixed zebrafish retina was probed with FRet 43, a monoclonal antibody that binds to double cone cells, and visualized using the ELF® 97 Immunohistochemistry Kit.

The yellow-green–fluorescent double cones stained with the ELF® 97 alcohol precipitate are oriented with the outer segments at the top of the stained configuration and the synaptic pedicles at the bottom. This section was counterstained with tetramethylrhodamine wheat germ agglutinin, which makes the rod outer segments (top left) and the inner plexiform layer and ganglion cell axons (bottom right) appear bright red. Wheat germ agglutinin also binds the region occupied by the cone outer segments and synaptic pedicles, which appear bright yellow because they are double-labeled with both the ELF® 97 alcohol precipitate and tetramethylrhodamine. Although this section has also been counterstained with Hoechst 33342, the blue-stained nuclei are barely visible in this photomicrograph. However, the double cones' inner fibers traverse the region occupied by the rod nuclei, and thus appear light blue. The inner segments, myoids, and nuclei of these double cone cells are labeled only with the ELF® 97 alcohol precipitate, giving them a characteristic green appearance. The image was obtained by triple exposure through optical filters appropriate for DAPI, tetramethylrhodamine, and the ELF® precipitate. Used with permission from J Histochem Cytochem 43:77 (1995).

What's New

The Scientist named the Applied Biosystems® Attune® Acoustic Focusing Cytometer one of the top 10 innovations of 2010. Here’s what the judges said:

“Designed to use sound waves to precisely control the movement of cells and increase instrument simplicity, sensitivity and throughput. Looks like it will be particularly useful for analyzing dilute cell samples. The simplicity and relatively low cost of the instrument should also increase the number of scientists who use flow cytometry.”

— H. Steven Wiley, Lead Biologist, Pacific NW National Labs (USA)

“With a footprint small enough to fit in a laminar-flow hood and a completely new approach to fluidics, the Attune cytometer promises less clogging than other flow cytometers, even while speeding through huge populations of cells.”

Check out the Invitrogen™ Protocols web area today. Here you will find detailed protocols for Invitrogen™, Molecular Probes®, Gibco®, and Dynal® products, and much more. Protocols for a broad range of research areas and applications are being added every day. Don’t see what you need? Request a protocol and provide feedback.